Methods for improving mechanical properties of a tissue or for regenerating an injured or diseased tissue

ABSTRACT

The present invention relates to enhancing mechanical properties of tissue such as collagenous or collagen-containing or elastin-containing tissue (e.g., tendons, ligaments, and cartilage) and treating related musculoskeletal and non-musculoskeletal conditions or injuries.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/810,021 filed on Feb. 25, 2019. The content of the application isincorporated herein by reference in its entirety.

GOVERNMENT INTERESTS

This invention was made with government support under AR072886 awardedby National Institutes of Health and CMMI-1560965 awarded by NationalScience Foundation. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to a tissue engineering and regenerativemedicine. Specifically, the present disclosure involves enhancingmechanical properties of tissue containing collagen or elastin such astendons, ligaments, cartilage, heart, lung, and skin and treatingrelated musculoskeletal or non-musculoskeletal conditions or injuries.

BACKGROUND OF THE INVENTION

Musculoskeletal conditions or injuries represent a critical healthconcern. According to the American Academy of Orthopedic Surgeons, overone in four Americans have a musculoskeletal impairment, costing theUnited States close to $850 billion each year. It was estimated thatannually musculoskeletal-related conditions in the US account for 132million doctor visits, 29 million emergency department visits, and 15million hospital stays. There is a need for enhancing mechanicalproperties of tissue and thereby treating related musculoskeletalconditions or injuries.

SUMMARY OF INVENTION

This invention relates to enhancing mechanical properties of tissue viaincreasing lysyl oxidase activity or mechanical stimulation and treatingrelated musculoskeletal or non-musculoskeletal tissues (e.g., skin)conditions or injuries.

In one aspect, the invention features a method for (i) improving amechanical property of a tissue or a component thereof or (ii)regenerating an injured or diseased tissue or developing embryonic/fetaltissue or a component thereof or (iii) enhance mechanical properties ofa healthy tissue in a subject. The method comprises applying amechanical stimulation to the tissue or cells therein. In someembodiments, the method further comprises increasing a level of lysyloxidase (LOX) activity in the tissue.

In another aspect, the invention provides a method for (i) improving amechanical property of a tissue or a component thereof or (ii)regenerating an injured tissue or a component thereof in a subject. Themethod comprises increasing a level of LOX activity in the tissue. Insome embodiments, the method further comprises applying a mechanicalstimulation to the tissue or cells therein.

In the above-described methods, examples of the mechanical stimulationinclude a dynamic stimulation, a cyclic stimulation, a staticstimulation, a deformation, a tensile stimulation, a compressivestimulation, a torsion stimulation, a shear stimulation, substratestiffness, and a mechanical loading. The mechanical stimulation can beindependent, or the mechanical stimulation can be combined with othertypes of treatments as described herein. One or more of the mechanicalstimuli can also be combined. The mechanical loading can comprise astatic loading, a dynamic loading, a cyclic loading, a compression,shear, torsion, or deformation.

The tissue mentioned above can be any tissue of interest. Examples ofthe tissue include, but not limited to, a healthy or normal tissue, aninjured tissue, a diseased tissue, an aging tissue, an adult tissue, ora developing embryonic/fetal tissue. The tissue or tissues can comprisea natural tissue, an engineered tissue, an embryonic tissue, a postnataltissue, a tissue in vitro, or a tissue in vivo. The mechanical propertyof the tissue can be one selected from the group consisting of elasticmodulus, tensile strength, torsional strength, elongation to break,hardness, compressive strength, burst strength, toughness, impactstrength, torsion, failure load, and stiffness.

In the above-described methods, the LOX activity can be an activity ofLOX or a LOX like (LOXL) protein, (e.g., LOX-like 1, LOX-like 2,LOX-like 3, or LOX-like 4). Increasing a level of LOX activity can becarried out by delivering to the tissue a LOX/LOXL enhancer. Examples ofthe enhancer include an agent selected from the group consisting of aLOX or LOXL (LOX/LOXL) polypeptide, a pre-pro LOX/LOXL polypeptide, apro LOX/LOXL polypeptide, a nucleic acid encoding one or more of saidpolypeptides, a viral particle having said nucleic acid, an engineeredcell expressing having said nucleic acid, bone morphogenetic protein-1(BMP-1), Fibronectin, Tolloid, Copper, Vitamin B6, Ascorbic acid, andProcollagen c proteinase.

In some embodiments, the methods described above can further compriseadministering a population of cells to the tissue. The cells can be (i)collagen-producing or elastin-producing cells or progenitor cellsthereof or (ii) engineered to release a specific factor that directly orindirectly promotes LOX/LOXL or pro-LOX/pro-LOXL gene expression,LOX/LOXL or pro-LOX/pro-LOXL protein expression, or LOX/LOXL enzymeactivity.

The invention further provides a pharmaceutical composition comprising(i) a LOX/LOXL enhancer, and (ii) a pharmaceutically acceptable carrieror excipient. Also provided is a kit comprising one or more of theLOX/LOXL enhancers and a packaging material.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objectives, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are a set of diagrams and photographs showingeffects of paralysis (decamethonium bromide (DMB) treatment) andhypermotility (4-aminopyridine (4-AP) treatment) on Hamburger-Hamiltonstage (HH)43 calcaneal tendons after 48 h (N≥5). (a) DMB treatment ledto lower elastic modulus than saline treatment. 4-AP treatment led tohigher elastic modulus than saline treatment. (b) Normalized fibrillarcollagen content did not change with DMB treatment or 4-AP treatmentcompared to saline controls. (c) Representative images of fibrillarcollagen detected by forward second harmonic generation (F-SHG) forsaline, DMB, 4-AP, 4-AP+β-aminopropionitrile (BAPN), and BAPNtreatments. (Scale bar: 10 mm; *p<, 0.05).

FIGS. 2A, 2B, 2C, 2D, and 2E are a set of diagrams showing LOX andLOXL1-4 exhibited distinct gene expression profiles in developingcalcaneal tendons (N≥3). (a) LOX mRNA expression levels of HH41 to hHH45tendons were higher than that of HH39. (b) LOXL1 mRNA maintainedrelatively constant levels until decreasing at HH45. (c) LOXL2 levelsdecreased from HH38 to HH45. (d) LOXL3 levels did not change from HH38to HH45. (e) LOXL4 mRNA levels decreased from HH38 to HH39 and thenremained constant to HH45. (*p<0.05; **p<0.01; †0.05<p<0.08).

FIGS. 3A and 3B are a set of diagrams showing that ProLOX and LOX:activity levels increased in calcaneal tendons during development (N≥5).(a) ProLOX levels increased from HH38 to HH42 and then plateaued. (b)LOX activity levels were constant from HH38 to HH42 and then increasedfrom HH42 to HH45. (*p<0.05; **p<0.01; †0.05<p<0.08).

FIGS. 4A and 4B are a set of diagrams showing DMB and 4-AP effects onLOX activity levels of HH43 calcaneal tendons after 48 h (N≥3). (a)Paralytic agents DMB and pancuronium bromide (PB) treatment eachdecreased LOX activity levels. 4-AP treatment had no effect on LOXactivity levels. (b) DMB treatment of isolated leg explants in vitro hadno effect on LOX activity levels. (*p<0.05; **p<0.01).

FIG. 5 is a diagram showing calcaneal tendon elastic moduli of HH43chick embryos treated for 48 h (N=5). BAPN treatment decreased modulusrelative to saline controls (same saline control data as in FIG. 1A);4-AP treatment increased modulus relative to saline controls (same dataas in FIG. 1 a ); 4-AP+BAPN treatment reduced modulus compared with 4-APtreatment alone. (*p<0.05, **p<0.01).

FIGS. 6A, 6B, 6C, 6D, 6E, and 6F are a set of photographs showingembryos were injected at HH43, harvested after 48 h, and staged on thebasis of anatomical features in comparison with non-injected HH45embryos (N≥6). Representative legs from (a) non-injected HH45 embryo;and (b) saline-treated; (c) DMB-treated, with hyperextended digits under‘rigid paralysis’; (d) 4-AP-treated.; (e) 4-AP+BAPN-treated; and (f)BAPN-treated HH43 embryos. (Scale bar: 10 mm; *p<0.05).

FIG. 7 is a diagram showing that treatment with an exogenous recombinantLOX (rLOX) increased elastic modulus of HH40 explant calcaneal tendonsrelative to saline controls. Data points for two rLOX treatments werecombined together to have N=3 for the rLOX-treated group.

FIG. 8 is a diagram showing effects of paralysis (no movement), inducedby either DMB or PB treatment, on density of collagen crosslinkerhydroxylysyl pyridinoline (HP).

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to tissue engineering, regenerative medicine,physical rehabilitation, or enhancement of various tissues. It involvesenhancing mechanical properties of tissue (such as tendons, ligaments,and cartilage), improving tissue mechanical properties, and treatingrelated musculoskeletal conditions or injuries. As disclosed herein, thetissue engineering can include artificially imposing mechanicalstimulation to engineered tissues or to explant tissues—which can thenbe implanted. Regenerative medicine via physical rehabilitation canutilize physical therapy in the form of passive movement ormuscle-generated forces to mechanically stimulate tissue(s) to treatconditions or injuries. In some embodiments, massage could be used tomechanically stimulate tissues (deep tissues, e.g., muscle; or skin;etc.).

Tissue Mechanical Property and Mechanical Loading

Certain aspects of this invention are based, at least in part, on anunexpected discovery that embryo movements regulate mechanical propertydevelopment of tissues, such as tendon.

Tendons transmit forces from muscles to bones to enable skeletalmotility. During development, tendons begin to bear load at the onset ofembryo movements. Using the chick embryo model, study disclosed hereinshowed that altered embryo movement frequency led to changes in elasticmodulus of calcaneal tendon. In particular, paralysis led to decreasedmodulus, whereas hypermotility led to increased modulus. Paralysis alsoled to reductions in activity levels of LOX, an enzyme forcrosslinking-mediated elaboration of tendon mechanical properties.Additionally, inhibition of LOX activity abrogated hypermotility-inducedincreases in modulus. Taken together, the findings disclosed hereinsuggest embryo movements are critical for tendon mechanical propertydevelopment and implicate LOX in this process. These findings expandcurrent knowledge of how functional tendons form during development andcould guide future clinical approaches to treat tendon defectsassociated with abnormal mechanical loading in utero.

Tendons are load-bearing collagenous tissues that enable skeletalmotility. Kicking depends heavily on calcaneal tendons to transmitforces from the calf muscle to the calcaneal bone. Embryo movements,such as kicking, have been implicated as critical regulators ofmusculoskeletal tissue development (1-8). Deprivation of movement viatreatment with DMB results in abnormal development of bone, meniscus,and joint (1, 3, 4). In contrast, 4-AP treatment to increase chickembryo motility increased bone growth (5, 9). Fewer studies have focusedon mechanical regulation of tendon development. Specifically, paralysisof early stage (e.g., HH24 and HH28) chick embryos with DMB leads toabnormal tendon marker expression patterns and tendon tissue morphologycompared to controls (3, 6). While these studies implicate movement as aregulator of tendon development, the influence of mechanical loading onthe formation of a mechanically functional tendon had not beeninvestigated.

With atomic force microscopy (AFM), non-linear increases in embryoniccalcaneal tendon modulus from HH28 to HH43 (˜day 5.5 to 17) werecharacterized. Interestingly, the most dramatic increases in tendonmodulus occur during stages that coincide with heightened movementactivity (frequency). In particular, kicking frequencies peak at HH40and HH43, the same stages when chick embryo calcaneal tendon modulusincreases dramatically. For reference, the chick embryo is near fullterm by HH45 (hatches at day 21), when the tendon is nearly ready tofunction as a load-bearing tissue during postnatal activities (walking,jumping, etc.). In the examples described below, study was carried outto examine how embryo movement frequency influences mechanical propertydevelopment during these critical stages of functional tendon formation.

The findings disclosed herein provide evidence that mechanical cues arecritical for embryonic tendon development. In particular, frequency ofmovements, such as kicking, can significantly influence tendonmechanical property elaboration and skeletal development. Additionally,the findings indicate LOX as a key player in this process. Thisinformation impacts clinical approaches to treat musculoskeletalabnormalities that result from aberrant embryonic or fetal movements inutero. In addition, mechanics in tendon development can be used toinform tissue engineering (e.g., tendon) and regeneration strategies.

Improving Mechanical Property

In some embodiments, the present disclosure provides methods forimproving mechanical property of a tissue (e.g., tendon, ligament, andcartilage). As disclosed herein, the methods can lead to an increase incollagen crosslinking, which leads to an increase in mechanical property(e.g., modulus). Accordingly, collagenous tissue produced using methodsof the present disclosure is extensively cross-linked and has enhancedmechanical properties, such as high(er) elastic modulus and othersdisclosed herein. Specifically, the present disclosure involves anenzyme-mediated collagen-crosslinking process to produce tissue such astendon, ligament, or cartilage, and to enhance its mechanicalproperties, maturation, and integration.

In some embodiments, this invention provides methods for improvingengineered tissues. An example of a tissue suitable for the methods ofthe present disclosure is an elastin-containing tissue or acollagen-containing, or collagenous, tissue, such as tendon, ligament,and cartilage. Collagenous tissue contains an intricate architecture ofcollagen crosslinks, as well as a variety of other components. As thiscollagen network structure and associated properties is inherent tonative collagenous tissues, the tissue engineering disclosed herein canbe used to produce tissues that mimic or are superior to native tissues.

Accordingly, this invention relates to methods of producing collagenoustissue possessing a high tensile strength involving treating connectivetissue cells under conditions effective for formation of enzyme-mediatedcollagen cross-links to produce collagenous tissue possessing a hightensile strength. Conditions effective for formation of enzyme-mediatedcollagen cross-links to produce a collagenous tissue having a hightensile strength may comprise application of applying a mechanicalstimulation and/or a LOX enhancer.

Mechanical Load

Cells, such as chondrocytes in cartilage, fibrochondrocytes in menisci,or tenocytes and fibroblasts in tendon and ligament, are able to altertheir metabolic activity in response to applied mechanical loads. Boththe level of strain applied and the dynamic frequency are important indetermining this response. These processes are believed to be majorfactors in determining cellular activity in these tissues. Themechanisms by which cells detect and respond to mechanical load aretermed mechanotransduction pathways and are complex and poorlyunderstood. Mechanotransduction events may be resolved intoextracellular components including cell deformation, hydrostaticpressures and streaming potentials, followed by intracellular signalingevents such as intracellular calcium fluxes, cAMP production andcytoskeletal alterations which finally lead to altered effector cellresponse.

The findings disclosed herein provide evidence that mechanical cues arecritical for embryonic tendon development and that LOX is a key playerin this process. The mechanics in tendon development can be used fortissue engineering and regeneration.

Some embodiments of the present invention may induce alterations in cellbehavior in response to mechanical loading through many of theaforementioned mechanotransduction pathways. Mechanical stimulation orloading may be applied to cells in situ within a target tissue.Alternatively, a mechanical stimulation or loading may be applied to acell-containing implant/construct prior to implantation to the targettissue using specially designed mechanical stimulation or applied to thecells within the construct post implantation through defined exerciseregimes or through externally applied regimes such as continuous passivemotion regimes. Mechanical stimulation or loading regimes, applied in astatic or dynamic manner may take a variety of forms including, e.g.,uniaxial compression or tension or hydrostatic pressure. Static anddynamic peak strain amplitude may be in the range between 0.5-30% (e.g.,1-25%, 5-20%, whilst dynamic frequencies should range from 0-10 Hz(e.g., 0.01-5 Hz, 0.1-3 Hz).

In some embodiments, exposure to a mechanical stimulation or loading canbe achieved by consistently applying strain (i.e., applying a force thatcauses a change in length) or by applying stress (i.e., applying amechanical load) to the tissue or implant over a predetermined period oftime. Alternatively, mechanical stimulation or loading can be achievedby applying stress or strain to the tissue or implant in a cyclicalpattern, for example, applying stress or strain for 1 minute every 5minutes over a total time of 48 hours. The stress or strain can beconstant throughout the time period, or alternatively, the stress orstrain can vary. The amount of stress or strain can vary, for example,from 5% to 15% using an appropriate device, such as a load cell. Thestress or strain can be applied in a uniaxial or multiaxial direction.The amount of strain applied can be measured in the cell culture systemsby any means known in the art, for example, by laser measurements usinglaser beams and deflection times. Means for applying mechanicalstimulation or loading are known in the art. Examples are described inUS20180216057, US20010016772, US20050025838, and US20040067833, whichare incorporated by reference in their entireties.

Mechanical stimulation or loading can be used together with a LOX. Insome embodiments, a LOX can be in its active form, while in others LOXcan be in an inactive form (e.g., a pro-enzyme or pre-pro-enzyme).Conditions effective for formation of enzyme-mediated collagencross-links to produce a collagenous or elastogenic tissue having a hightensile strength may comprise exogenous application of one or moreadditional enhancers described herein and/or additional enzymes (such asglucose oxidase and catalase). Conditions effective for the formation ofenzyme-mediated collagen cross-links to produce a collagenous tissuehaving a high tensile strength may comprise culturing connective tissuecells under hypoxic conditions. In some embodiments, the level ofenzyme-mediated collagen-crosslinks is directly proportional to theconcentration of exogenously-supplied or endogenously expressed LOXpresent during the treating step.

In some embodiments, a collagenous tissue treated with a method of thisinvention possesses a high tensile strength and can have an elasticmodulus value higher (e.g., at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10-fold, or more) than the elastic modulus value of a controlcollagenous tissue.

The methods and compositions described herein may find use as intreating diseases and syndromes related to collagen or elastincrosslinking deficiency (e.g., osteolathyrism, Ehlers Danlos syndromeType IV, etc.).

Lysyl Oxidase and Lysyl Oxidase-Like (LOXL) Proteins

Lysyl oxidase is an extracellular copper-dependent enzyme that catalyzesformation of aldehydes from lysine residues in collagen and elastinprecursors. LOX catalyzes oxidative deamination of peptidyl lysine andhydroxylysine residues in collagens, and peptidyl lysine residues inelastin. The resulting peptidyl aldehydes spontaneously condense andundergo oxidation reactions to form the lysine-derived covalentcross-links required for the normal structural integrity of theextracellular matrix. In the reaction of lysyl oxidase with itssubstrates, hydrogen peroxide and ammonium are released in quantitiesstoichiometric with the peptidyl aldehyde product. See, e.g., Kagan etal., J. Cell. Biochem. 88:660-72 (2003).

Lysyl oxidase is secreted into the extracellular environment where it isthen processed by proteolytic cleavage to a functional 30 (kilo daltons)kDa enzyme and an 18 kDa propeptide. The 30 kDa lysyl oxidase isenzymatically active whereas the 50 kDa proenzyme is not. ProcollagenC-proteinases process pro-lysyl oxidase to its active form and areproducts of the Bmpl, TII1 and TII2 genes. The localization of theenzyme is mainly extracellular, although processed lysyl oxidase alsolocalizes intracellularly and nuclearly. Sequence coding for thepropeptide is moderately (60-70%) conserved among LOX and the LOXLproteins, whereas the sequence coding for the C-terminal 30 kDa regionof the proenzyme in which the active site is located is highly conserved(approximately 95%). See Kagan et al., J. Cell Biochem. 59:329-38(1995).

Five different lysyl oxidases are known to exist in both humans andmice, LOX and four LOX related, or LOX-like proteins (LOXL1, LOXL2,LOXL3, and LOXL4). LOX and the LOX-like proteins are referred tocollectively as “LOX/LOXL” or “lysyl oxidase type enzymes” for thepurposes of the present disclosure. The five forms of lysyl oxidasesreside on five different chromosomes. These family members show someoverlap in structure and function, but appear to have distinct functionsas well. For example, although the main activity of LOX is the oxidationof specific lysine residues in collagen and elastin outside of the cell,it may also act intracellularly, where it may regulate gene expression.In addition, LOX induces chemotaxis of monocytes, fibroblasts and smoothmuscle cells. Further, a deletion of LOX in knockout mice appears to belethal at parturition (Hornstra et al., J. Biol. Chem. 278:14387-14393(2003)), whereas LOXL deficiency causes no severe developmentalphenotype (Bronson et al., Neurosci. Lett. 390:118-122 (2005)).

The main activity of LOX is the oxidation of specific lysine residues incollagen and elastin outside of the cell, however, it may also actintracellularly, where it may regulate gene expression (Li et al., Proc.Natl. Acad. Sci. USA 94:12817-12822 (1997), Giampuzzi et al., J. Biol.Chem. 275:36341-36349 (2000)). In addition, LOX induces chemotaxis ofmonocytes, fibroblasts and smooth muscle cells (Lazarus et al., MatrixBiol. 14:727-731 (1995), Nelson et al., Proc. Soc. Exp. Biol. Med.188:346-352 (1988)). LOX itself is induced by a number of growth factorsand steroids such as TGF-β, TNF-α and interferon (Csiszar, Prog. Nucl.Acid Res. 70:1-32 (2001)).

As used herein, the term “lysyl oxidase” refers to an enzyme thatcatalyzes the following reaction:peptidyl-L-lysyl-peptide+O₂+H₂O→peptidyl-allysyl-peptide+NH₃+H₂O₂. Othersynonyms for lysyl oxidase (EC 1.4.3.13) include protein-lysine6-oxidase and protein-L-lysine: oxygen 6-oxidoreductase (deaminating).See, e.g., Harris et al., Biochim. Biophys. Acta 341:332-44 (1974);Rayton et al., J. Biol. Chem. 254:621-26 (1979); Stassen, Biophys. Acta438:49-60 (1976). A copper-containing quinoprotein with a lysyl adductof tyrosyl quinone at its active center, LOX catalyzes the oxidation ofpeptidyl lysine to result in the formation of peptidylalpha-aminoadipic-delta-semialdehyde. Once formed, this semialdehyde canspontaneously condense with neighboring aldehydes or with other lysylgroups to form intra- and interchain cross-links. See, e.g., Rucker etal., Am. J. Clin. Nutr. 67:996S-1002S (1998).

An example of lysyl oxidase or lysyl oxidase-like protein include theenzyme having an amino acid sequence substantially identical to apolypeptide expressed or translated from one of the following sequences:EMBL/GenBank accession numbers: M94054; AAA59525.1; 545875; AAB23549.1;578694; AAB21243.1; AF03929 I; AAD02130.1; BC074820; AAH74820.1;BC074872; AAH74872.1; M84150; and AAA59541.1. Additional examplesinclude those described in e.g., US20180155447, US20060029588, andWO2014065863.

LOX has highly conserved protein domains, conserved in several speciesincluding human, mouse, rat, chicken, fish and Drosophila. Shown below a417 amino acid (aa) human lysyl oxidase (hLOX, AAA59525.1, SEQ ID NO:1). The human LOX family has a highly conserved C-terminal regioncontaining the 205 amino acid residues (SEQ ID NO: 3) and in particularthe aa 283-aa 417 (SEQ ID NO: 4, in bold in the sequence shown below)LOX catalytic domain. See e.g., Bhuvanasundar et al. Bioinformation.2014 Jul. 22; 10(7):406-12 and Csiszar, Prog Nucleic Acid Res Mol Biol.2001; 70:1-32, both of which are incorporated herein by reference intheir entireties. The conserved aa 283-aa 417 region contains the copperbinding (Cu), conserved cytokine receptor like domain (CRL), and thelysyl-tyrosylquinone cofactor site (LTQ). The predicted extracellularsignal sequences are known in the art (See e.g., U.S. 20180155447, whichis incorporated herein by reference in its entirety). Twelve cysteineresidues are also similarly conserved, wherein two of them reside withinthe prepropeptide region and ten are in the catalytically activeprocessed form of LOX (Csiszar, Prog. Nucl. Acid Res. 70:1-32 (2001)).The conserved region also includes a fibronectin binding domain.

One embodiment of LOX is human lysyl oxidase (hLOX, AAA59525.1, 417 aa):

(SEQ ID NO: 1) MRFAWTVLLLGPLQLCALVHC APPAAGQQQPPREPPAAPGAWRQQIQWENNGQVFSLLSLGSQYQPQRRRDPGAAVPGAANASAQQPRTPILLIRDNRTAAARTRTAGSSGVTAGRPRPTARHWFQAGYSTSRARERGASRAENQTAPGE VPALSNLRPPSRVDGMVGDDPYNPYKYSDDNPYYNYYDTYERPRPGGRYRPGYGTGYFQYGLPDLVADPYYIQASTYVQKMSMYNLRCAAEENCLASTAYRADVRDYDHRVLLRFPQRVKNQGTSDFLPSRPRYSWEWHSCHQHYHSMDEFSHYDLLDANTQRRVAEGHKASFCLEDTSCDYGYHRRFACTAHTQGLSPGCYDTYGADIDCQWIDITDVKPGNYILKVSVNPSYLVPESDYTNNVVRCDI RYTGHHAYASGCTISPY

The prepropeptide region of this hLOX contains the signal peptide(underlined, SEQ ID NO: 2), and is cleaved, the cleavage site predictedto be between Cys21-Ala22 (bold above), to generate a signal sequencepeptide and a 48 kDa amino acid propeptide form of LOX, which is stillinactive. The propeptide is N-glycosylated during passage through theGolgi that is secreted into the extracellular environment where theproenzyme, or propeptide, is cleaved between Glyl68-Asp169 (bold above)by a metalloendoprotease, a procollagen C-proteinase, which are productsof the Bmpl, TII1 and TII2 genes. BMP I (bone morphogenetic protein I)is a procollagen C-proteinase that processes the propeptide to yield afunctional 30 kDa enzyme and an 18 kDa propeptide. The sequence codingfor the pro-peptide is moderately (60-70%) conserved, whereas thesequence coding for the C-terminal 30 kDa region (underlined, SEQ ID NO:3) of the proenzyme in which the active site is located is highlyconserved (approximately 95%). (Kagan and Li, J. Cell. Biochem.88:660-672 (2003); Kagan et al., J. Cell Biochem. 59:329-38 (1995)). TheN-glycosyl units are also subsequently removed. LOX occurs inunprocessed and/or processed (mature) forms. The mature form of LOX istypically active although, in some embodiments, unprocessed LOX is alsoactive.

Particular examples of a LOXL enzyme or protein are described in Molnaret al., Biochim Biophys Acta. 1647:220-24 (2003); Csiszar, Prog. Nucl.Acid Res. 70:1-32 (2001); and in WO01/83702, all of which are hereinincorporated by reference in their entirety. In the present invention,“LOXL” refers to a lysyl oxidase-like protein in general. These enzymesinclude LOXL1, encoded by mRNA deposited at GenBank/EMBL BC015090;AAH15090.1; LOXL2, encoded by mRNA deposited at GenBank/EMBL U89942;LOXL3, encoded by mRNA deposited at GenBank/EMBL AF282619; AAK51671.1;and LOXL4, encoded by mRNA deposited at GenBank/EMBL AF338441;AAK71934.1.

Shown below is an exemplary human LOXL1 sequence, GenBank Acc. No.AAH15090

(SEQ ID NO: 5) 1 malargsrql galvwgaclc vlvhgqqaqp gqgsdparwrqliqwenngq vysllnsgse 61 yvpagpqrse sssrvllaga pqaqqrrshg sprrrqapslplpgrvgsdt vrgqarhpfg 121 fgqvpdnwre vavgdstgma rartsysqqr hggsassysasafastyrqq psypqqfpyp 181 qapfvsqyen ydpasrtydq gfvyyrpagg gvgagaaavasagviypyqp raryeeyggg 241 eelpeyppqg fypaperpyv pppppppdgl drryshslysegtpgfeqay pdpgpeaaqa 301 hggdprlgwy ppyanpppea ygppralepp ylpvrssdtpppggerngaq qgrlsvgsvy 361 rpnqngrglp dlvpdpnyvq astyvqrahl yslrcaaeekclastayape atdydvrvll 421 rfpqrvknqg tadflpnrpr htwewhschq hyhsmdefshydlldaatgk kvaeghkasf 481 cledstcdfg nlkryactsh tqglspgcyd tynadidcqwiditdvqpgn yilkvhvnpk 541 yivlesdftn nvvrcnihyt gryvsatnck ivqs

Similar potential signal peptides as those described above for LOX havebeen predicted at the amino terminus of LOXL, LOXL2, LOXL3, and LOXL4.The predicted signal cleavage sites are between Gly25-Gln26 for LOXL,between Ala25-Gln26, for LOXL2, and between Gly25-Ser26 for LOXL3. Theconsensus for BMP-1 cleavage in pro-collagens and pro-LOX is betweenAla/Gly-Asp, and often followed by an acidic or charged residue. Apotential cleavage site to generate active LOXL is Gly303-Asp304,however, it is then followed by an atypical Pro. LOXL3 also has apotential cleavage site at Gly447-Asp448, which is followed by an Asp,processing at this site may yield an active peptide of similar size toactive LOX. A potential cleavage site of BMP-I was also identifiedwithin LOXL4, at residues Ala569-Asp570 (Kim et al., J. Biol. Chem.278:52071-52074 (2003)). LOXL2 may also be proteolytically cleavedanalogously to the other members of the LOXL family and secreted (Akiriet al., Cancer Res. 63:1657-1666 (2003)).

The terms “LOX” and “LOXL” also encompass functional fragments orderivatives that substantially retain enzymatic activity catalyzing thedeamination of lysyl residues. Typically, a functional fragment orderivative retains at least 50% of 60%, 70%, 80%, 90%, 95%, 99% or 100%of its lysyl oxidation activity. It is also intended that a LOX/LOXLprotein can include conservative amino acid substitutions that do notsubstantially alter its activity. Suitable conservative substitutions ofamino acids are known to those of skill in this art and may be madegenerally without altering the biological activity of the resultingmolecule. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity. See, e.g., Watson, et al.,Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. Co., p. 224. Conservative and non-conservative amino acidsubstitutions have been described above.

As used herein, the term “conservative sequence modifications” refers toamino acid modifications that do not significantly affect or alter theactivity of a LOX or LOXL. Conservative amino acid substitutions areones in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art.

Amino acid substitutions can be made, in some cases, by selectingsubstitutions that do not differ significantly in their effect onmaintaining (a) the structure of the peptide backbone in the area of thesubstitution, (b) the charge or hydrophobicity of the molecule at thetarget sit; or (c) the bulk of the side chain. For example, naturallyoccurring residues can be divided into groups based on side-chainproperties; (1) hydrophobic amino acids (norleucine, methionine,alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic aminoacids (cysteine, serine, threonine, asparagine, and glutamine); (3)acidic amino acids (aspartic acid and glutamic acid); (4) basic aminoacids (histidine, lysine, and arginine); (5) amino acids that influencechain orientation (glycine and proline); and (6) aromatic amino acids(tryptophan, tyrosine, and phenylalanine). Substitutions made withinthese groups can be considered conservative substitutions. Examples ofsubstitutions include, without limitation, substitution of valine foralanine, lysine for arginine, glutamine for asparagine, glutamic acidfor aspartic acid, serine for cysteine, asparagine for glutamine,aspartic acid for glutamic acid, proline for glycine, arginine forhistidine, leucine for isoleucine, isoleucine for leucine, arginine forlysine, leucine for methionine, leucine for phenylalanine, glycine forproline, threonine for serine, serine for threonine, tyrosine fortryptophan, phenylalanine for tyrosine, and/or leucine for valine.Exemplary substitutions are shown in the table below. Amino acidsubstitutions may be introduced into human LOX/LOXL and the productsscreened for retention of the biological activity of human LOX/LOXL.

Original Residue Exemplary Substitutions Ala (A) Val; Leu; Ile Arg (R)Lys; Gln; Asn Asn (N) Gln; His; Asp, Lys; Arg Asp (D) Glu; Asn Cys (C)Ser; Ala Gln (Q) Asn; Glu Glu (E) Asp; Gln Gly (G) Ala His (H) Asn; Gln;Lys; Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Lys (K) Arg; Gln; Asn Met (M) Leu; Phe; Ile Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ser (S) Thr Thr (T) Val;Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe; Thr; Ser Val (V) Ile; Leu; Met;Phe; Ala; Norleucine

A major regulator of tendon mechanical properties (10, 12), LOXoxidatively deaminates lysine and hydroxylysine residues of collagenmolecules to facilitate crosslink formation between adjacent collagenmolecules (13). In addition, LOX also affects elastin to crosslinkelastin. As shown in this invention, inhibition of LOX activity viaβ-aminopropionitrile (BAPN) reduced both LOX-mediated crosslink densityand tendon modulus during tissue formation without affecting collagencontent or organization. Notably, statistical analysis showed moduluscorrelated with crosslink density (r²=0.80, p<0.0001) with and withoutperturbation of LOX activity (12).

Furthermore, study was carried out to understand the role of embryomovement in regulating mechanical property development of calcanealtendons and whether LOX is involved in this process. It was hypothesizedthat perturbation of chick embryo movement activity during laterdevelopmental stages alters LOX activity and that this leads to changesin tendon mechanical properties. Paralysis (zero movement frequency) andhypermotility (increased movement frequency) led to decreases andincreases in elastic modulus, respectively. Furthermore, LOX activitywas downregulated with paralysis, and inhibition of LOX duringhypermotility abrogated modulus increases that occurred withhypermotility alone. Taken together, mechanical loading of tendon duringdevelopment is critical for the development of mechanical properties andmay require the involvement of LOX. The findings disclosed hereinestablish new insights into the role of embryo movement inmusculoskeletal tissue development and how the LOX enzyme may beinvolved in mechanical regulation of tendon development.

The chick embryo is a well-established model to study development ofmusculoskeletal tissues, sharing significant similarities with mammals(10, 12, 20-27). Notably, the LOX amino acid sequence is highlyconserved across human, mouse, rat, and chicken (16). Additionally,unlike mammals, the chick embryo can be manipulated in ovo andindependently of maternal influences. Injecting DMB and 4-AP into theair pocket likely affected various tissues throughout the chick embryo.The multiple perturbations in the study disclosed herein collectivelyand compellingly suggest that altered embryo movement frequency was themajor contributor to changes in elastic modulus of the calcanealtendons. Localizing drug treatments to only the leg and testingdifferent DMB and 4-AP dosages as well as different kicking frequenciescan be carried out. Complementary studies using an in vitro bioreactorsystem (28-30) could impose more highly controlled strains, strainrates, and frequencies on explant tendons.

Cells

Collagen-producing cells, elastin-producing cells, and/or progenitorcells thereof can be employed in this invention, which providescollagens or elastin or both for the integrated growth, differentiation,and regeneration of tissue capable of substantially functioning as,e.g., tendon, ligament, cartilage, or bone. The cells can beadministered directly to a site of an injured tissue or be part of animplant or construct to be implanted. By forming an integrated constructin the shape of a tissue (e.g., a ligament or a joint) outside the body,the implant can adhere to the surface of the ligament or joint andintegrate appropriately. As would be readily apparent to one of skill inthe art, progenitor cell types, such as mesenchymal stem cells, primarychondrocytes, or osteoblasts are useful for these applications.

The cells can be autologous, allogeneic or xenogeneic with respect to ahost, preferably the cells are autologous. Conveniently, examples of thecells can be chondrocytes, fibrochondrocytes, fibroblasts, osteoblasts,or sub-populations thereof, which have a differentiated phenotype.Alternatively, precursors of the aforementioned cell types may be usedwhich have the potential to differentiate into such cells.

In some embodiments, the cells used in conjunction with the methods ofthe present disclosure may be derived from mesenchymal, embryonic,induced pluripotent stem cells, skin cells, or other stem cells. Thecells may be derived from any source and site for obtaining a cellsample comprising a sufficient number of cells to produce a collagenoustissue. Such cells and cell samples may be obtained by any meanssuitable for obtaining a cell sample comprising a sufficient number ofcells. In certain embodiments, such a means may comprise enzymaticdigestion of native tissue. Suitable enzymes for such an enzymaticdigestion include, but are not limited to, one or more collagenases.

The presently disclosed implantable composition can comprise one or morecells that can develop into a suitable replacement of a target tissue(e.g., tendon, ligament, cartilage, or bone). Particularly, the one ormore cells comprise, or are derived from, a precursor cells, such as butnot limited to a stem cell.

As used herein, the term “stem cell” refers to any unipotent,multipotent, pluripotent and/or totipotent cell that can bedifferentiated into a desired lineage. As such, the presently disclosedsubject matter can employ stem cells that can be differentiated into atissue appropriate for replacement of native pathological tissues.Representative stem cells include embryonic stem (ES) cells, embryonicgerm (EG) cells (e.g., pluripotent cells derived from primordial germcells), and somatic stem cells (alternatively referred to herein as“adult stem cells”).

In some embodiments, the one or more cells described herein comprise anadult stem cell. Adult stem cells can be derived from various adulttissues including, but not limited to liver, bone marrow, umbilical cordblood, brain, peripheral blood, blood vessels, skeletal muscle, adiposetissue, and skin. Methods for the isolation, culturing, and manipulationof adult stem cells from various sources can be found in U.S. Pat. Nos.6,242,252 and 6,872,389 (hepatic stem cells); U.S. Pat. No. 6,387,367(hematopoietic/mesenchymal stem cells); Kogler et al. (2004) J Exp Med200:123-135 (placental cord blood); Williams et al. (1999) The AmericanSurgeon 65:22-26 (skeletal muscle); U.S. Pat. No. 6,777,231 (adiposetissue); and Blanpain et al. (2004) Cell 118:635-648 (skin), the entirecontents of each of which are hereby incorporated in their entireties.

Representative techniques for deriving, growing, and manipulating EScells and EG cells are disclosed in the following publications: Evansand Kaufman (1981) Nature 292:154-156; Martin (1981) Proc Natl Acad SciUSA 78:7634+7638; Robertson (1986) Trends Genet 2:9-13; PCTInternational Patent Application Publications WO 96/22362; WO 97/32033;and WO 98/43679; and U.S. Pat. Nos. 6,200,806; 6,090,622; 5,843,780;5,690,926; 5,670,372; and 5,453,357; and references therein, all ofwhich are incorporated by reference herein in their entireties.

In some embodiments, the cells can be genetically modified, e.g., toexpress exogenous genes or to repress the expression of endogenousgenes. In some embodiments, the present invention provides methods ofgenetically modifying such cells and populations. In accordance withthese methods, the cells can be exposed to an expression constructcomprising a nucleic acid including a transgene, such that the nucleicacid is introduced into the cell under conditions appropriate for thetransgene to be expressed within the cell. The transgene generally is anexpression cassette, including a coding polynucleotide operably linkedto a suitable promoter. The coding polynucleotide can encode a protein,or it can encode a biologically active (e.g., functional) fragment of aprotein.

Thus, for example, the coding polynucleotide can encode a LOX/LOXL, agene conferring resistance to a toxin, a hormone (such as peptide growthhormones, hormone releasing factors, sex hormones, adrenocorticotrophichormones, cytokines (e.g., interferins, interleukins, lymphokines),etc.), a cell-surface-bound intracellular signaling moiety (e.g., celladhesion molecules, hormone receptors, etc.), a factor promoting a givenlineage of differentiation, etc. Of course, where it is desired toemploy gene transfer technology to deliver a given transgene, thesequence will be known. In some embodiments, the coding polynucleotideencodes a growth factor. In some embodiments, the coding polynucleotideencodes a LOX/LOXL, a LOX/LOXL enhancer (e.g., BMP-1) or a functionalfragment thereof.

The cells can be stably or transiently transfected or transduced with anucleic acid of interest using a plasmid, viral or alternative vectorstrategy. With respect to the cells, nucleic acids of interest include,but are not limited to, those encoding gene products which enhance theproduction of extracellular matrix components found in tendon, ligament,or cartilage, such as collagen, TGF-β, BMP, activin and insulin-likegrowth factor.

Thus, in some embodiments, the transduction of regulatory genes into thecells, for example stem cells, can be performed with viral vectors(adenovirus, retrovirus, adeno-associated virus, or other vector)purified by cesium chloride banding or any other well-known method at amultiplicity of infection (viral units:cell) of between 10:1 to 2000:1.Cells can be exposed to the virus in serum-free or serum-containingmedium in the absence or presence of a cationic detergent such aspolyethyleneimine or LIPOFECTAMINE (INVITROGEN, Carlsbad, Calif., UnitedStates of America) for a period of 1 hour to 24 hours (Byk et al. (1998)Human Gene Therapy 9:2493-2502; Sommer et al. (1999) Calcif. Tissue Int.64:45-49) or in three-dimensional cultures by incorporation of theplasmid DNA vectors directly into a biocompatible polymer (Bonadio etal. (1999) Nat. Med. 5:753-759).

In some embodiments, cells, for example stem cells, are transfected withthe gene to be expressed to produce cells having stably incorporatedtherein the DNA encoding the molecules to be expressed. Stabletransfections can be obtained by culturing and selecting for expressionof the desired encoded molecules. In some embodiments, the cells thatexhibit stable expression can be seeded onto or into the appropriatefiber matrix and implanted in a subject. For the tracking and detectionof functional proteins encoded by these genes, the viral or plasmid DNAvectors can contain a readily detectable marker gene, such as the greenfluorescent protein (GFP) or β-galactosidase enzyme, both of which canbe tracked by histochemical means.

Within the expression cassette, the coding polynucleotide can beoperably linked to a suitable promoter. Examples of suitable promotersinclude prokaryotic promoters and viral promoters (e.g., retroviralinverted terminal repeats (ITRs), long terminal repeats (LTRs),immediate early viral promoters (IEp), such as herpes virus IEp (e.g.,ICP4-IEp and ICP0-IEp), cytomegalovirus (CMV) IEp, and other viralpromoters, such as Rous Sarcoma Virus (RSV) promoters, and MurineLeukemia Virus (MLV) promoters). Other suitable promoters are eukaryoticpromoters, such as enhancers (e.g., the rabbit β-globin regulatoryelements), constitutively active promoters (e.g., the β-actin promoter,etc.), signal specific promoters (e.g., inducible promoters such as apromoter responsive to RU486, etc.), and tissue-specific promoters. Itis well within the skill of the art to select a promoter suitable fordriving gene expression in a predefined cellular context. The expressioncassette can include more than one coding polynucleotide, and it caninclude other elements (e.g., polyadenylation sequences, sequencesencoding a membrane-insertion signal or a secretion leader, ribosomeentry sequences, transcriptional regulatory elements (e.g., enhancers,silencers, etc.), and the like), as desired.

The expression cassette containing the transgene can be incorporatedinto a genetic vector suitable for delivering the transgene to thecells. Depending on the desired end application, any such vector can beso employed to genetically modify the cells (e.g., plasmids, naked DNA,viruses such as adenovirus, adeno-associated virus, herpes viruses,lentiviruses, papillomaviruses, retroviruses, etc.). Any method ofconstructing the desired expression cassette within such vectors can beemployed, many of which are well known in the art (e.g., direct cloning,homologous recombination, etc.). Of course, the choice of vector willlargely determine the method used to introduce the vector into the cells(e.g., by protoplast fusion, calcium-phosphate precipitation, gene gun,electroporation, infection with viral vectors, etc.), which aregenerally known in the art.

In some embodiments, the genetically altered cells can be employed asbioreactors for producing the product of the transgene. In someembodiments, the genetically modified cells are employed to deliver thetransgene and its product to a subject. For example, the cells, oncegenetically modified, can be introduced into the subject underconditions sufficient for the transgene to be expressed in vivo.

Thus, the present invention also provides in some embodiments methodsfor treatment of tendon, ligament or joint disorders. Disclosed hereinare approaches for regenerating collagen-containing tissues. Furtherdisclosed herein is the use of progenitor, stem, or primary cells inconjunction with a composition that comprises a medium capable ofsupporting the growth and differentiation of the cells into functionaltissue, but not necessarily recapitulating the native structure of thetissue.

Methods of Treatment and Prevention/Regenerating Injured Tissue orComponent Thereof

The presently disclosed methods involve the regeneration of tissue,promotion of healing of an injury (e.g., tendon or ligament injury), orenhancing healthy tissues. As disclosed herein, the methods can be usedfor treatment of birth defects in utero too. To that end, anembryo/fetus in need of such a treatment can be stimulated to increaseone or more types of mechanical stimulation (e.g., movement-induced orother), and the LOX activity could be enhanced to improve mechanicalproperties of developing tissues.

The regeneration of tissue refers to the process of renewal and growthof cells and extracellular matrix components within a particular tissuethat results in the production of tissue that has a cellular componentand architecture that allows for the normal functions of the particulartissue type.

An injury or wound refers to damage or harm to a structure or functionof the body caused by intrinsic and/or extrinsic factors. Non-limitingintrinsic or extrinsic factors that can cause an injury or wound includethose of chemical, mechanical, thermal, bacterial, or physical means andencompass those that occur as the result of surgical procedures,overuse, or environmental conditions. The wound can be an open wound inwhich the skin is broken (e.g., lacerations, abrasions, puncture wound)or a closed wound. Particular wounds that can be treated by thisinvention include, but are not limited to, tendon or ligament injuries,bone injuries (e.g., complete or partial fractures), skin wounds, andskeletal muscle injuries.

Intrinsic factors that can contribute to the development of injuries totendons and/or ligaments include genetic susceptibility, overuse, poorbiomechanics, poor nutrition, and obesity. The extrinsic factors areoften related to sports and include excessive forces or loading, poortraining techniques, environmental conditions, and surgical procedures.The injury to the tendon and/or ligament can be a closed wound or anopen wound, where the skin is lacerated, cut or punctured. The injurycan include inflammation, a sprain, strain, tearing, stretching, orlaceration of the tendon or ligament.

A tendon is a band of connective tissue that connects muscles to bonesor cartilage. A ligament is a band of connective tissue that connectsbones to other bones to form joints. Injuries to tendons includetendinitis (acute tendon injury accompanied by inflammation), tendinosis(chronic tendon injury with degeneration at the cellular level and noinflammation), and other tendinopathies exhibiting chronic tendon injurywith no etiological implications. With tendinosis, damage to collagen,cells, and the vascular components of the tendon can occur, such asirregularities of collagen fibrils (e.g., disorientation, degeneration,thinning, non-uniformity in length or diameter, increase in the amountof glycosaminoglycans between the fibrils), rounded tenocytes or othercell abnormalities, and the ingrowth of blood vessels.

The healing of an injury to any type of tendon can be promoted with theinvention disclosed herein, including a hand flexor tendon, a tendonwithin the rotator cuff, and an Achilles tendon, and within horses, asuperficial digital flexor tendon (SDFT) and a deep digital flexortendon (DDFT) of either the hindlimb(s) or forelimb(s).

Likewise, the healing of an injury to any type of ligament can bepromoted with the invention disclosed herein, including an anteriorcruciate ligament (ACL), posterior cruciate ligament (PCL), lateralcollateral ligament (LCL), medial collateral ligament (MCL), and inhorses, a suspensory ligament of either the hindlimb(s) or forelimb(s).A common ligament injury in horses that can be healed according to thepresently disclosed methods is proximal suspensory desmitis, aninflammation of the suspensory ligament just below the hock.

In some embodiments the invention includes both prophylactic andtherapeutic methods of (i) improving a mechanical property of a tissueor a component thereof or (ii) regenerating an injured tissue or acomponent thereof in a subject.

Subjects having or at risk for the condition can be identified by, forexample, any one or a combination of known in the art. Administration ofa prophylactic agent can occur prior to the manifestation of symptomscharacteristic of the condition or decrease, such that a condition isprevented or, alternatively, delayed in its progression. Depending onthe type of the condition, for example, a LOX/LOXL enhancer can be usedfor treating the subject.

In one aspect, the method includes increasing a level of LOX activity inthe tissue by e.g., administering to the subject a LOX/LOXL enhancer,e.g., a LOX or LOXL (e.g., a LOX or LOXL polypeptide, or active fragmentthereof, or a nucleic acid encoding the polypeptide or active fragmentthereof), or another agent that modulates (i.e., increases) LOX or LOXLexpression or at least one LOXL activity.

As used herein, an active fragment of a LOX or LOXL polypeptide retainsthe ability to oxidize lysine residues in elastin and collagen. Forexample, an active fragment can be missing a portion of the N-terminalsequence but retaining the C-terminal enzymatic domain (e.g., a fragmentcomprising from amino acids about 145, 179, 326, 338, to about 574,referring to the human sequence, GenBank Acc. No. AAH15090, SEQ ID NO:5). An active fragments can include the C-terminal 28 kD of thepolypeptide. Fragments can be generated by recombinant DNA techniquesknown in the art, or can be produced by enzymatic digestion of all orpart of a full length LOX or LOXL polypeptide, e.g., digestion with bonemorphogenetic protein-1 (BMP-1; see Borel et al., supra). Such fragmentscan comprise the N-terminal portion of the sequence that is conservedbetween LOX, LOXL1, LOXL2, LOXL3, and LOXL4, e.g., comprising theputative copper-binding site, the lysine tyrosylquinone cofactorformation, and/or the cytokine receptor-like domain (see, e.g., Maki,Dissertation: LYSYL OXIDASES, Cloning and Characterization of the Fourthand the Fifth Human Lysyl Oxidase Isoenzymes, and the Consequences of aTargeted Inactivation of the First Described Lysyl Oxidase Isoenzyme inMice, Collagen Research Unit, Biocenter Oulu and Department of MedicalBiochemistry and Molecular Biology, University of Oulu (2002)). Smalleror larger fragments can also be used.

In some embodiments, to modulate LOX or LOXL expression or activity(e.g., for therapeutic purposes), a cell is contacted with a LOX or LOXLnucleic acid or polypeptide (or active fragment thereof), or an agentthat modulates one or more of the activities of LOX or LOXL polypeptideactivity associated with the cell. An agent that modulates LOX or LOXLpolypeptide activity can be, e.g., an agent as described herein, such asa nucleic acid or a polypeptide, a naturally-occurring binding partnerof a LOX or LOXL polypeptide, a LOXL antibody, a LOXL agonist, apeptidomimetic of a LOXL agonist, or other small molecule. The agent canbe synthetic, or naturally occurring. The cell can be an isolated cell,e.g., a cell removed from a subject or a cultured cell, or can be a cellin situ in a subject.

A LOX/LOXL enhancer agent can, in some embodiments, stimulate one ormore LOX/LOXL activities. Examples of such stimulatory agents includeactive LOX/LOXL polypeptide or an active fragment thereof, and a nucleicacid molecule encoding a LOX/LOXL polypeptide or active fragmentthereof.

As defined herein, a therapeutically effective amount of a LOX/LOXLnucleic acid or polypeptide composition is a dosage effective to treator prevent a particular condition for which it is administered. The dosewill depend on the composition selected, i.e., a polypeptide or nucleicacid. The compositions can be administered from one or more times perday to one or more times per week, including once every other day. Theskilled artisan will appreciate that certain factors may influence thedosage and timing required to effectively treat a subject, including butnot limited to the severity of the condition, previous treatments, thegeneral health and/or age of the subject, and other conditions present.Moreover, treatment of a subject with a therapeutically effective amountof the therapeutic LOX/LOXL compositions of the invention can include asingle treatment or a series of treatments, as well as multiple (i.e.,recurring) series of treatments.

Dosage, toxicity and therapeutic efficacy of such LOX/LOXL compositionscan be determined by pharmaceutical procedures known in the art in cellcultures or experimental animals, e.g., for determining the LD50 (thedose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Compositions that exhibit hightherapeutic indices are preferred. While compositions that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compositions locally to the site of affectedtissue to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Pharmaceutical Compositions and Methods of Administration

The invention further provides pharmaceutical compositions comprising aLOX/LOXL enhancer, for use in the treatment of conditions mentionedabove. Such compositions typically include a LOX/LOXL enhancer, and apharmaceutically acceptable carrier. A LOX/LOXL enhancer or activatorrefers to (i) an agent that stimulates one or more LOX/LOXL activitiesor (ii) a positive modulator of LOX/LOXL activity or expression The termcovers agents (such as BMP-1 and Tolloid) that cleave ProLox to producean active mature LOX. As used herein, examples of a LOX/LOXL enhancercan be a LOX/LOXL nucleic acid or polypeptide (or active fragmentthereof) or other positive modulator of LOX/LOXL activity or expression,e.g., a LOX polypeptide, a pre-proLOX polypeptide, a proLOX polypeptide,a nucleic acid encoding said LOX/pre-proLOX polypeptide/proLOXpolypeptide, a viral particle having said nucleic acid, an engineeredcell expressing said LOX, pre-proLOX, or proLOX polypeptide, BMP-1,Fibronectin, Tolloid, Copper, Vitamin B6, Ascorbic acid, and Procollagenc proteinase.

As used herein the language “pharmaceutically acceptable carrier”includes saline, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

A LOX/LOXL enhancer pharmaceutical composition is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral (e.g., intravenous, intradermal, orsubcutaneous), oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

LOX/LOXL enhancer pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. For intravenousadministration, suitable carriers include physiological saline,bacteriostatic water, CREMOPHOR (BASF, Parsippany, N.J.) or phosphatebuffered saline (PBS). In all cases, the composition must be sterile andshould be fluid to the extent that easy syringability exists. It shouldbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activeLOX/LOXL enhancer in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle, whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, typical methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral LOX/LOXL enhancer compositions generally include an inert diluentor an edible carrier. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules, e.g., gelatincapsules. Oral compositions can also be prepared using a fluid carrier.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the LOX/LOXL enhancer can be deliveredin the form of an aerosol spray from pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer. Such methods include those described in U.S.Pat. No. 6,468,798.

Systemic administration of a LOX/LOXL enhancer can also be bytransmucosal or transdermal means. For transmucosal or transdermaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart, and include, for example, for transmucosal administration,detergents, bile salts, and fusidic acid derivatives. Transmucosaladministration can be accomplished through the use of nasal sprays orsuppositories. For transdermal administration, the active compounds areformulated into ointments, salves, gels, or creams as generally known inthe art.

In some embodiments, compositions comprising a LOX/LOXL enhancer fortransdermal application can further comprise cosmetically acceptablecarriers or vehicles and any optional components. A number of suchcosmetically acceptable carriers, vehicles and optional components areknown in the art and include carriers and vehicles suitable forapplication to skin (e.g., sunscreens, creams, milks, lotions, masks,serums, etc.), see, e.g., U.S. Pat. Nos. 6,645,512 and 6,641,824. Inparticular, optional components that may be desirable include, but arenot limited to absorbents, anti-acne actives, anti-caking agents,anti-cellulite agents, anti-foaming agents, anti-fungal actives,anti-inflammatory actives, anti-microbial actives, anti-oxidants,antiperspirant/deodorant actives, anti-skin atrophy actives, anti-viralagents, anti-wrinkle actives, artificial tanning agents andaccelerators, astringents, barrier repair agents, binders, bufferingagents, bulking agents, chelating agents, colorants, dyes, enzymes,essential oils, film formers, flavors, fragrances, humectants,hydrocolloids, light diffusers, nail enamels, opacifying agents, opticalbrighteners, optical modifiers, particulates, perfumes, pH adjusters,sequestering agents, skin conditioners/moisturizers, skin feelmodifiers, skin protectants, skin sensates, skin treating agents, skinexfoliating agents, skin lightening agents, skin soothing and/or healingagents, skin thickeners, sunscreen actives, topical anesthetics, vitamincompounds, and combinations thereof.

The LOX/LOXL enhancer compositions can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal or vaginaldelivery. Such suppositories can be used particularly for the treatmentof conditions associated with the loss of in elastic fibers that affectthe pelvic organs, e.g., pelvic organ prolapse and/or urinaryincontinence, inter alia.

LOX/LOXL enhancer compositions comprising nucleic acids can also beadministered by any method suitable for administration of nucleic acidagents. These methods include gene guns, bio injectors, and skin patchesas well as needle-free methods such as the micro-particle DNA vaccinetechnology disclosed in U.S. Pat. No. 6,194,389, and the mammaliantransdermal needle-free vaccination with powder-form vaccine asdisclosed in U.S. Pat. No. 6,168,587. Additionally, intranasal deliveryis possible, as described in, inter alia, Hamajima et al., Clin.Immunol. Immunopathol. 88(2), 205-10 (1998). Liposomes (e.g., asdescribed in U.S. Pat. No. 6,472,375) and microencapsulation can also beused. Biodegradable targetable microparticle delivery systems can alsobe used (e.g., as described in U.S. Pat. No. 6,471,996).

In one embodiment, LOX/LOXL enhancer compositions are prepared withcarriers that will protect against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using techniques known in the art. Thematerials can also be obtained commercially, e.g., from Alza Corporationand Nova Pharmaceuticals, Inc. Liposomal suspensions (includingliposomes targeted to specific cells with monoclonal antibodies) canalso be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration for thetreatment or prevention of a condition as mentioned above.

Administration of the compositions described above can be carried out byany method known to one of ordinary skill in the art. In someembodiments, suitable methods for administration of the compositions ofthe presently disclosed subject matter include, but are not limited toinjection into the target tissue or target site. The term “targettissue” as used herein refers to an intended site for engraftmentfollowing administration to a subject.

In some embodiments, the compositions comprise cells present in a matrix(e.g., a gel) within the pores of a fiber scaffold. The fiber scaffoldcan be implanted at a pre-determined site (i.e., a ligament, a tendon,or a joint) to replace, repair, and/or restore a target tissue and/orstructure at the particular site of insertion. In some embodiments, thefiber scaffold can be implanted in a subject to alleviate tissue loss,damage, injury, or combinations thereof.

The fiber scaffolds can be implanted into the subject at the site inneed of treatment using standard surgical techniques. In someembodiments, the fiber scaffold is constructed, seeded with cells andcultured in vitro prior to implantation. The cells can be cultured inthe device, tested for viability, and then implanted. In someembodiments, the fiber scaffold is constructed, seeded with cells andcultured in vivo after or during implantation. In some embodiments, thescaffold is implanted without cells. In some embodiments, the fiberscaffolds can be used for delivery of multiple different cell types. Thescaffold can be implanted in one or more different areas of the body tosuit a desired application.

In addition, there are situations where it could be desirable to usemore than one matrix, each implanted at the most optimum time for growthof the attached cells to form a functioning three-dimensional structurefrom the different matrices.

An effective dose of a composition of the presently disclosed subjectmatter is administered to a subject in need thereof. An “effectiveamount” or a “therapeutic amount” is an amount of a therapeuticcomposition sufficient to produce a biologically or clinically relevantresponse in a subject being treated. The actual amount of a therapeuticagent in the composition can be varied so as to achieve the desiredtherapeutic response for a particular subject. The selected dosage levelwill depend upon several factors including, but not limited to theability of the cells or their progeny to engraft the target tissue, theroute of administration, combination with other drugs or treatments, theseverity of the condition being treated, and the condition and priormedical history of the subject being treated.

The potency of a composition can vary, and therefore an “effectiveamount” can vary. However, using standard assay methods, one skilled inthe art can readily assess the potency and efficacy, and adjust thetherapeutic regimen accordingly. In view of the disclosure of thepresent invention, one of ordinary skill in the art can tailor thedosages to an individual subject, taking into account the particularformulation, method of administration to be used with the composition,and particular disease treated. Further calculations of dose canconsider subject height and weight, severity and stage of symptoms, andthe presence of additional deleterious physical conditions. Suchadjustments or variations, as well as evaluation of when and how to makesuch adjustments or variations, are well known to those of ordinaryskill in the art of medicine.

The subjects treated in the present invention are in some embodimentshuman subjects, although it is to be understood that the presentlydisclosed subject matter is effective with respect to all vertebrateanimals, including mammals, which are intended to be included in theterm “subject”. Moreover, a mammal is understood to include anymammalian species in which treatment or prevention of a disease isdesirable, particularly agricultural and domestic mammalian species.

Kits

The disclosure also provides kits, where the kits include one or morecomponents employed in methods of the invention, e.g., LOX/LOXLenhancers (such as a LOX polypeptide, a pre-proLOX polypeptide, a proLOXpolypeptide, a nucleic acid encoding said LOX, pre-proLOX, or proLOXpolypeptide, a viral particle having said nucleic acid, an engineeredcell expressing said LOX, pre-proLOX, or proLOX polypeptide, BMP-1,Fibronectin, Tolloid, Copper, Vitamin B6, Ascorbic acid, and Procollagenc proteinase), vectors, and cells, as described herein. Kits may alsoinclude tubes, buffers, packaging materials, etc., and instructions foruse. The various reagent components of the kits may be present inseparate containers, or some or all of them may be pre-combined into areagent mixture in a single container, as desired.

In addition to the above components, the kits may further includeinstructions for practicing the subject methods. These instructions maybe present in the kits in a variety of forms, one or more of which maybe present in the kit. One form in which these instructions may bepresent is as printed information on a suitable medium or substrate,e.g., a piece or pieces of paper on which the information is printed, inthe packaging of the kit, in a package insert, etc. Yet another form ofthese instructions is a computer readable medium, e.g., diskette,compact disk (CD), hard drive etc., on which the information has beenrecorded. Yet another form of these instructions that may be present isa website address which may be used via the internet to access theinformation at a removed site.

Definitions

As used herein, “mechanical stimulation or loading” shall mean forcesapplied to a structure or a component which are mechanical in nature, ora mechanical force. In one aspect, the mechanical loading can becompression. In another aspect, the mechanical loading can be tension.

The term “collagen” as used herein refers to a group of naturallyoccurring proteins found in the flesh and in connective tissues ofmammals. It is the main component of connective tissue, and is the mostabundant protein in mammals, making up about 25% to 35% of thewhole-body protein content. Collagen, in the form of elongated fibrils,is mostly found in fibrous tissues, such as tendon, ligament, and skin,and is also abundant in cornea, cartilage, bone, blood vessels, the gut,and intervertebral disc. So far, 29 types of collagen have beenidentified and over 90% of the collagen in the body is of type I (skin,tendon, vascular, ligature, organs, bone), type II (cartilage), type III(reticulate (main component of reticular fibers), and type IV (whichforms the bases of cell base membrane).

As used herein, “treatment” means the application or administration of aLOX or LOXL1 enhancer therapeutic agent to a subject (e.g., a human or aveterinary or experimental animal subject), or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a subject, who has a condition, a symptom of condition, or apredisposition to get a condition, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, prevent, oraffect the disease, symptoms of the condition, or the predisposition toget the condition. A LOX or LOXL1 enhancer therapeutic agent includes,but is not limited to, small molecules including peptidomimetics,peptoids, nucleic acids (e.g., nucleic acids encoding a LOX or LOXLpolypeptide or active fragment thereof), aptamers, carbohydrates,polysaccharides, non-nucleic acid small organic molecules, inorganicmolecules, polypeptides, antibodies, ribozymes, and drugs.

The terms “peptide,” “polypeptide,” and “protein” are used hereininterchangeably to describe the arrangement of amino acid residues in apolymer. A peptide, polypeptide, or protein can be composed of thestandard 20 naturally occurring amino acid, in addition to rare aminoacids and synthetic amino acid analogs. They can be any chain of aminoacids, regardless of length or post-translational modification (forexample, glycosylation or phosphorylation).

A conservative modification or functional equivalent of a peptide,polypeptide, or protein disclosed in this invention refers to apolypeptide derivative of the peptide, polypeptide, or protein, e.g., aprotein having one or more point mutations, insertions, deletions,truncations, a fusion protein, or a combination thereof. It retainssubstantially the activity to of the parent peptide, polypeptide, orprotein (such as those disclosed in this invention). In general, aconservative modification or functional equivalent is at least 60%(e.g., any number between 60% and 100%, inclusive, e.g., 60%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to a parent (e.g.,SEQ ID NO: 1, 3, 4, or 5).

A nucleic acid or polynucleotide refers to a DNA molecule (e.g., a cDNAor genomic DNA), an RNA molecule (e.g., an mRNA), or a DNA or RNAanalog. A DNA or RNA analog can be synthesized from nucleotide analogs.The nucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated nucleic acid” refers toa nucleic acid the structure of which is not identical to that of anynaturally occurring nucleic acid or to that of any fragment of anaturally occurring genomic nucleic acid. The term therefore covers, forexample, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. The nucleic acid described above can be used toexpress the protein of this invention. For this purpose, one canoperatively linked the nucleic acid to suitable regulatory sequences togenerate an expression vector.

A vector refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. The vector can becapable of autonomous replication or integrate into a host DNA. Examplesof the vector include a plasmid, cosmid, or viral vector. The vectorincludes a nucleic acid in a form suitable for expression of the nucleicacid in a host cell. Preferably the vector includes one or moreregulatory sequences operatively linked to the nucleic acid sequence tobe expressed.

A “regulatory sequence” includes promoters, enhancers, and otherexpression control elements (e.g., polyadenylation signals). Regulatorysequences include those that direct constitutive expression of anucleotide sequence, as well as tissue-specific regulatory and/orinducible sequences. The design of the expression vector can depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein or RNA desired, and the like. The expressionvector can be introduced into host cells to produce a polypeptide ofthis invention. A promoter is defined as a DNA sequence that directs RNApolymerase to bind to DNA and initiate RNA synthesis. A strong promoteris one which causes mRNAs to be initiated at high frequency.

The term “operably-linked” or “operably-linked” is used herein to referto an arrangement of flanking sequences wherein the flanking sequencesso described are configured or assembled so as to perform their usualfunction. Thus, a flanking sequence operably linked to a coding sequencemay be capable of effecting the replication, transcription and/ortranslation of the coding sequence. For example, a coding sequence isoperably linked to a promoter when the promoter is capable of directingtranscription of that coding sequence. A flanking sequence need not becontiguous with the coding sequence, so long as it functions correctly.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence, andthe promoter sequence can still be considered “operably-linked” to thecoding sequence. Each nucleotide sequence coding for a polypeptide willtypically have its own operably linked promoter sequence.

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, which may include a promoter operably linkedto the nucleotide sequence of interest that may be operably linked totermination signals. The coding region usually codes for a functionalRNA of interest. The expression cassette including the nucleotidesequence of interest may be chimeric. The expression cassette may alsobe one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. The expression ofthe nucleotide sequence in the expression cassette may be under thecontrol of a constitutive promoter or of a regulatable promoter thatinitiates transcription only when the host cell is exposed to someparticular stimulus. In the case of a multicellular organism, thepromoter can also be specific to a particular tissue or organ or stageof development.

Such expression cassettes can include a transcriptional initiationregion linked to a nucleotide sequence of interest. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the gene of interest to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselectable marker genes.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence. It may constitute an “uninterrupted codingsequence”, i.e., lacking an intron, such as in a cDNA, or it may includeone or more introns bounded by appropriate splice junctions.

As used herein, the percent homology between two amino acid sequences isequivalent to the percent identity between the two sequences. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, known in the art.

The percent identity between two amino acid sequences can be determinedusing the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci.,4:11-17 (1988)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4. In addition, the percent identity betweentwo amino acid sequences can be determined using the Needleman andWunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

As used herein, “treating” or “treatment” refers to administration of acompound or agent to a subject who has a disorder or is at risk ofdeveloping the disorder with the purpose to cure, alleviate, relieve,remedy, delay the onset of, prevent, or ameliorate the disorder, thesymptom of the disorder, the disease state secondary to the disorder, orthe predisposition toward the disorder. The terms “prevent,”“preventing,” “prevention,” “prophylactic treatment” and the like referto reducing the probability of developing a disorder or condition in asubject, who does not have, but is at risk of or susceptible todeveloping a disorder or condition.

An effective amount refers to the amount of an active compound/agentthat is required to confer a therapeutic effect on a treated subject.Effective doses will vary, as recognized by those skilled in the art,depending on the types of conditions treated, route of administration,excipient usage, and the possibility of co-usage with other therapeutictreatment.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo.

A “pharmaceutically acceptable carrier,” after administered to or upon asubject, does not cause undesirable physiological effects. The carrierin the pharmaceutical composition must be “acceptable” also in the sensethat it is compatible with the active ingredient and can be capable ofstabilizing it. One or more solubilizing agents can be utilized aspharmaceutical carriers for delivery of an active compound. Examples ofa pharmaceutically acceptable carrier include, but are not limited to,biocompatible vehicles, adjuvants, additives, and diluents to achieve acomposition usable as a dosage form. Examples of other carriers includecolloidal silicon oxide, magnesium stearate, cellulose, and sodiumlauryl sulfate.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human mammals, non-human primates (particularly higher primates),dog, rodent (e.g., mouse or rat), guinea pig, cat, and rabbit, andnon-mammals, such as birds, amphibians, reptiles, etc. In oneembodiment, the subject is a human. In another embodiment, the subjectis an experimental, non-human animal or animal suitable as a diseasemodel.

Musculoskeletal conditions, diseases and disorders are abnormalconditions of muscles and their associated ligaments, tendons,connective tissues and bones. The causes of musculoskeletal conditionsmay include, but are not limited to, wear and tear from dailyactivities, trauma to an area, auto accidents, falls, fractures,sprains, dislocations, direct blows to muscles, postural strain,repetitive movements, overuse and prolonged immobilization and diseaserelated conditions. Examples include an injury or pain in the body'sbones, joints, ligaments, muscles, tendons, nerves, tendons, cartilagesand structures that support limbs, neck and back, which is adegenerative disease and inflammatory condition that causes pain andimpair normal activities. Examples of specific musculoskeletal disordersinclude all diseases related to bones, joints, ligaments, muscles,nerves, tendons, cartilages and structures that support limbs, neck andback. The musculoskeletal disorder can be selected from the groupconsisting of sprains, strains and tears of ligaments, tendons, musclesand cartilage, tendonitis, tenosynovitis, fibromyalgia, osteoarthritis,rheumatoid arthritis, polymyalgia rheumatica, bursitis, acute andchronic back pain, osteoporosis, carpal tunnel syndrome, DeQuervains'sdisease, trigger finger, tennis elbow, rotator cuff, ganglion cysts,osteogenesis imperfecta, Duschennes, Hurler's and Hunter's syndromes andcombination thereof.

As used herein, the term “mesenchymal stem cells” or “MSCs” refers tomultipotent stem cells, which can differentiate into a variety of celltypes, including for example, osteoblasts, chondrocytes and adipocytesetc. The mesenchymal stem cells or MSCs may be derived from any tissuesources, including but not limited to bone marrow tissues, adiposetissue, muscle tissue, corneal stroma or dental pulp of deciduous babyteeth, umbilical cord tissues or umbilical cord blood etc. In oneexample of the invention, the MSCs are bone marrow MSCs.

As disclosed herein, a number of ranges of values are provided. It isunderstood that each intervening value, to the tenth of the unit of thelower limit, unless the context clearly dictates otherwise, between theupper and lower limits of that range is also specifically disclosed.Each smaller range between any stated value or intervening value in astated range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included or excluded in therange, and each range where either, neither, or both limits are includedin the smaller ranges is also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

The term “about” generally refers to plus or minus 10% of the indicatednumber. For example, “about 10%” may indicate a range of 9% to 11%, and“about 1” may mean from 0.9-1.1. Other meanings of “about” may beapparent from the context, such as rounding off, so, for example “about1” may also mean from 0.5 to 1.4.

EXAMPLES Example 1

This example describes material and methods used in Examples 2-5 below.All materials and reagents were obtained from THERMOFISHER (MA, USA)unless otherwise noted.

Chick Embryo Culture and Injections

Fertilized white leghorn eggs (University of Connecticut poultry farm)were incubated at 37° C. under high humidity. For developmentalcharacterizations, eggs were not injected. For other experiments, asingle dose of sterile saline (control), 0.2% DMB (rigid paralysis),0.2% pancuronium bromide (PB) (flaccid paralysis), 0.2% 4-AP(hypermotility), or 0.2% 4-AP and 5 mg BAPN per gram of embryo dry mass(to simultaneously induce hypermotility and inhibition of LOX activity)was injected at the air sac end of an HH43 egg and incubated for another48 h, as previously described (10). DMB dosage was based on previousstudies that paralyzed the embryo without affecting gross development(3). 4-AP dosage was based on previous studies that showed a singleinjection induced hypermotility up to 400% in HH36 to HH44 chick embryosfor at least 24 h (14). At specific timepoints, chick embryos weresacrificed by decapitation and staged based on anatomical features (15).The calcaneal tendon was homogenized in TRIzol LS reagent, snap frozenfor protein and enzyme assays, or embedded for cryosectioning.

Leg Explant Culture

Lower limbs were isolated from anatomically staged HH43 chick embryosand cultured in growth medium (Dulbecco's Modified Eagle's Medium, 10%fetal bovine serum, 1% antibiotic/antimycotic) supplemented with salineor 0.2% DMB. Media were changed after 24 h. Calcaneal tendons wereharvested for LOX activity assay after 48 h.

Total RNA Isolation and Reverse Transcription Polymerase Chain Reaction(RT-PCR)

Total RNA was extracted from TRIZOL LS-preserved samples and RT-PCR wasperformed using SUPERSCRIPT III One-Step RT-PCR System with Platinum TaqHigh Fidelity DNA Polymerase (INVITROGEN, CA) with primer pairs for LOX,LOX-like (LOXL) 1 through 4, and 18S (Table 1). Product bands in the gelwere analyzed using fluorescence intensity-based densitometryquantification with IMAGEJ (NIH, Bethesda, Md.).

TABLE 1 Primer sequences. SEQ SEQ ID ID Gene Forward NO: Reverse NO: LOXTCGGGCGGATGTTAGAGACT 6 AGCTGGCGTCTAACAAGTCA 7 LOXL1 TGCTACGACACCTACAACGC8 GTGGTTTTGGGCTCATGGTG 9 LOXL2 CAATTCCTTGCATCCCCAACC 10TAGGGCCAGGAATGCTCAGA 11 LOXL3 AGTTGGCACACTCGTACCG 12CATCTTCACACCAACGACATCCT 13 LOXL4 TGCGATGATGGCTTCGACTT 14CTGGCCGTAAGTAGCACTGT 15 18S AACGGGGCCATGATTAAGAGG 16TTGCGCCGGTCCAAGAATTT 17

Protein and Enzyme Activity Assays

Western blot to semi-quantitatively measure proLOX levels was performedwith rabbit anti-proLOX antibody or rabbit anti-β-actin antibody (ABCAM,MA). ProLOX and (β-actin levels were quantified by densitometry at ˜50kDa and ˜40 kDa molecular bands, respectively. LOX activity of eachsample was measured using a LOX activity assay kit (ABCAM, MA).Recombinant LOXL2 enzyme (R&D Systems, MN) was used as a positivecontrol for each assay.

Cryopreservation and Cryosectioning

Calcaneal tendons of staged embryos were cryoembedded as previouslydescribed (10) and cryosectioned at 50 μm thickness.

Two Photon Microscopy and Image Analysis

Tendon cryosections were immersed in Ca²⁺/Mg²⁺-free phosphate bufferedsaline (PBS) to remove O.C.T. A 20× water objective imaged a 220×220 μmregion in the center of tendon midsubstance in PBS. Collagen wascharacterized by F-SHG at 800 nm excitation, and signal was collected bya photomultiplier tube using a 405DF30 filter. Background was determinedwith laser off. Background was subtracted from each F-SHG image usingMATLAB (MATHWORKS, MA) and then average pixel intensities of each imagewere calculated to assess collagen content per unit area. Prior studyshowed that this measurement of collagen content correlated highly withhydroxyproline content measured with biochemical assays (12).

AFM

Force volume-AFM was used to characterize tendon elastic modulus usingpreviously established methods (10). Briefly, tendon cryosections wereimmersed in PBS to remove O.C.T. A silicon nitride tip probe (˜20 nmradius) with a spring constant of 0.06 N/m (BRUKER, CA) was used on anMFP-3D AFM (ASYLUM RESEARCH, CA) to obtain force curves in a 64×642-dimensional array over a 10 μm×10 μm tissue region in the center ofthe tendon midsubstance with a 1.0 μm indentation trigger point. Thesample was indented at 6 μm/s, at which tendon elastic properties werepreviously characterized with negligible viscous effects (10). Elasticmodulus was calculated by fitting force displacement curves at eachindentation to the Hertzian model using equations adapted from aprevious study (10).

Statistical Analysis

For RT-PCR, tendons from both legs each from at least three embryos(N≥3) were characterized. For western blot and LOX activity assays,tendons from both legs from at least three embryos (HH41 and earlier) ortwo embryos (HH42 and later) were pooled and five pools (N=5) werecharacterized. For two photon imaging and AFM, tendons from one leg eachfrom at least five embryos (N≥5) were characterized based on a previousstudy (10). F-SHG was used to image three non-overlapping regions (seeTwo photon microscopy and image analysis section above) in themidsubstance of each tendon. AFM was used to indent one region (see AFMsection above) with apparent tendon fibrillar structure in themidsubstance of each tendon. One-way ANOVA was performed for comparisonof LOX family mRNA levels, proLOX levels, LOX activity levels amongdifferent developmental stages, and average stages of saline-, DMB-,4-AP-, and 4-AP+BAPN-treated HH43 chick embryos at harvest after 48 h oftreatment (α=0.05). Tukey's post-hoc test was used to perform multiplecomparison analysis to evaluate the statistical differences between twospecific groups at the same timepoint. Two-sample t-test was performedto compare elastic moduli, collagen content, and LOX activity levelsbetween treatment conditions (α=0.05). All analyses were performed withGRAPHPAD PRISM v.7.0a (La Jolla, Calif.).

Example 2 Tendon Elastic Modulus was Affected by Embryo Movement

DMB treatment of HH43 chick embryos resulted in paralysis andsignificantly reduced calcaneal tendon modulus relative to controls(FIG. 1A). 4-AP treatment induced hypermotility and significantlyupregulated tendon modulus relative to controls (FIG. 1A). Neithertreatment affected collagen content or apparent organization relative tocontrols (FIGS. 1B and 1C).

Example 3 LOX Levels Increase During Development Whereas LOXL1-4 LevelsDo Not

LOX mRNA expression levels of HH41 through HH45 tendons were higher thanthat of HH39 (FIG. 2A). LOXL1 mRNA maintained relatively constant levelsuntil decreasing at HH45 (FIG. 2B). LOXL2 levels decreased from HH38through HH45 (FIG. 2C). LOXL3 mRNA levels showed no changes betweenstages (FIG. 2D). LOXL4 mRNA levels decreased from HH38 to HH39 and thenremained constant through HH45 (FIG. 2E).

ProLOX levels increased significantly from HH38 to HH42 and thenplateaued (FIG. 3A). LOX activity levels were constant from HH38 to HH42and then increased from HH42 through HH45 (FIG. 3B).

Example 4 LOX Activity Levels were Affected by Embryo Movement

DMB treatment of HH43 embryos significantly reduced LOX activity levelsin calcaneal tendons (FIG. 4A). PB treatment to induce flaccid paralysisresulted in calcaneal tendons with significantly lower LOX activitylevels than controls (FIG. 4A). In contrast, 4-AP treatment had noeffects on LOX activity levels compared to controls (FIG. 4A). LOXactivity levels of calcaneal tendons of leg explants cultured with DMBwere similar to controls (FIG. 4B).

Example 5 Perturbation of LOX Activity Abrogated Hypermotility-InducedIncreases in Modulus

4-AP treatment led to higher calcaneal tendon modulus than controls(FIG. 5 ). In contrast, 4-AP+BAPN treatment resulted in significantlylower modulus compared to 4-AP treatment alone, but was similar tocontrols. BAPN treatment alone reduced modulus relative to controls.

Example 6 Gross Development was Minimally Affected by Treatments

HH43 chick embryos treated with saline, DMB, 4-AP, and BAPN for 48 hexhibited similar anatomical features as non-injected HH45 chickembryos, reflecting normal development (Table 3). In contrast, chickembryos treated with 4-AP+BAPN staged to HH44 (Table 3). Grossly, legsharvested from saline-, 4-AP-, and 4-AP+BAPN-, and BAPN-treated embryosappeared similar to non-injected HH45 legs (FIGS. 6A, 6B, 6D, 6E, and6F). DMB-treated chick embryos were still in rigid paralysis andpossessed hyperextended digits at 48 h (FIG. 6C).

In the above examples, assays were carried out to test the hypothesisthat embryo movement frequency regulates tendon mechanical propertydevelopment, focusing on HH43, when the embryo has formed functionalmuscles and tendons and is kicking at the highest frequency duringdevelopment (11). HH43 chick embryos were treated with DMB to induceparalysis (zero frequency movement), saline (normal frequency movement),and 4-AP to induce hypermotility (high frequency movement). Elasticmodulus of calcaneal tendons decreased with paralysis and increased withhypermotility, relative to controls (FIG. 1A), demonstrating movementcan regulate the development of tendon mechanical properties. Inaddition, paralysis led to reductions in LOX activity levels (FIG. 4A),implicating LOX as an important player in normal movement-regulateddevelopment of tendon mechanical properties. Changes in LOX activitywere not detected after 48 h of hypermotility, however inhibition of LOXactivity during hypermotility abrogated the increases observed withhypermotility alone (FIG. 5 ), suggesting LOX may also be involved inmechanical upregulation of tendon mechanical properties. Taken together,these findings demonstrate movement critically regulates tendonmechanical property development and implicate LOX in this process.

These findings are significant because relatively little was known aboutthe role of mechanics in tendon development. In previous studies, DMBtreatment of HH24 chick embryos for 72 h diminished tendon markerexpression in forelimb zeugopod and digits (6). DMB-induced paralysis ofHH35 chick embryos also led to altered Tenascin-C protein distributionpatterns by HH39 (3). These studies showed that movement is importantfor tendon marker expression and patterning during earlier developmentalstages. However, the role of mechanics in regulating tendon developmentat later stages had not been studied.

(a) Tendon Elastic Modulus is Affected by Paralysis and Hypermotility

DMB irreversibly binds to acetylcholine receptors in the motor end plateto trigger an immediate and permanent contraction of the muscle (7).Consequently, DMB treatment induces a “rigid” phenotype of the lowerlimb, effectively imposing a static (zero frequency) load on thecalcaneal tendon. Here, mechanical testing of the calcaneal tendonsrevealed that paralysis reduced modulus by 2-fold relative to controls(FIG. 1A). To induce hypermotility, embryos were treated with 4-AP, apotassium channel blocker that prolongs depolarization at theneuromuscular junctions to stimulate continuous firing of musclecontraction (8). 4-AP treatment increased embryo movement frequency by200% within 1 min of injection, which lasted 48 h (data not shown).Mechanical testing of calcaneal tendons showed that 4-AP treatmentincreased modulus by 2-fold relative to controls (FIG. 1A).Collectively, these results suggest embryo movement is criticallyrequired for the normal development of tendon mechanical properties, andthat it is possible to enhance tendon mechanical properties byincreasing movement frequency.

(b) LOX Levels Increase During Development but LOXL1-4 Levels Do Not

Interestingly, collagen content and apparent collagen organization didnot change with either treatment relative to controls (FIGS. 1B and 1C).On the basis of these findings, it was asked whether LOX is involved inthe mechanically induced changes in modulus. LOX and LOXL family membershave been shown to exhibit similar catalytic functions (16). To examinewhich LOX family members may be involved in the development of embryonictendon mechanical properties, mRNA levels of LOX and LOXL family memberswere examined (FIG. 2). It was found that LOXL1 to 4 levels eitherremained constant or decreased during development. In contrast, LOXlevels increased beginning at HH39, peaked at HH42, and then plateauedthrough HH45. LOX was the only family member that increased duringdevelopment. Because elastic modulus also increases during development,LOX was further studied.

ProLOX and LOX activity levels each exhibited distinct stage-specifictrends, with dramatic increases at the latest stages. Interestingly,movement frequency increases in a stage-specific manner, and alsoincreases dramatically at the latest stages (11). Chick embryo bilaterallimb movement frequency peaks at HH43 (11). Coincidentally, LOX activitylevels increased significantly from HH42 to HH43 (FIG. 3B). Notably, LOXactivity levels (FIG. 3B) also correlated most highly with previouslyreported developing embryonic tendon moduli (10), (r²=0.97; p<0.05)(Table 2). Based on these data, it was hypothesized that movementregulates mechanical property development of calcaneal tendon, and thatthese events involve LOX.

TABLE 2 Pearson's correlation between previously reported elastic moduli(10) and LOX levels (mRNA, proLOX, LOX activity) for HH38 to HH43calcaneal tendons. Residual value ( r² ) p-value LOX mRNA vs. modulus0.56 0.25  proLOX vs. modulus 0.93 0.034 LOX activity vs. modulus 0.970.016

(c) Paralysis Downregulates LOX Activity

DMB treatment downregulated LOX activity levels of calcaneal tendons by2-fold compared to saline controls (p<0.05) (FIG. 4A), paralleling the2-fold decrease in modulus (FIG. 1A). To test whether this decrease inLOX activity levels was due specifically to rigid paralysis induced byDMB, flaccid paralysis was induced via PB treatment. PB-induced flaccidparalysis also led to 2-fold reductions in LOX activity levels relativeto controls (p<0.05) (FIG. 4A). Taken together, the two differentmethods to induce paralysis led to similar reductions in LOX activitylevels. To confirm these results were due to paralysis and not thechemical treatment itself, isolated legs were cultured ex ovo with DMB-or saline-supplemented growth medium for 48 h. No changes in tendon LOXactivity levels were detected (FIG. 4B), suggesting reductions in LOXactivity levels after paralysis were not due to biochemical effects ofDMB. These results strongly implicate LOX as an important player in hownormal embryo movement regulates the development of tendon mechanicalproperties.

Collagen content and organization of both DMB- and 4-AP-treated tendonsappeared normal despite decreases in modulus and LOX activity levels(FIGS. 1B and 1C). This was consistent with study that showed inhibitionof LOX activity decreases embryonic tendon moduli via reductions inLOX-mediated collagen crosslink density, and that this occurs withoutaffecting collagen content or organization (10, 12). Perhaps DMB-inducedparalysis led to decreases in LOX activity, which in turn reducedcollagen crosslinking, which then led to the decrease in modulus. Futurestudies could use mass spectrometry to measure changes in LOX-mediatedcrosslink density (12).

(d) Hypermotility-Induced Enhancement of Tendon Modulus May Involve LOX

LOX activity levels did not differ between 4-AP and saline treatments(FIG. 4A). A previous study with osteoblasts detected increases in LOXactivity at 18 h after treatment (17). Based on this, it is possiblethat the 48 h timepoint tested was too late, and missed a window of timeduring which LOX activity levels were higher. To test the potentialinvolvement of LOX with an alternative approach, embryos were treatedwith 4-AP+BAPN to induce hyperactivity and inhibit LOX activitysimultaneously. Strikingly, 4-AP+BAPN treatment led to reductions incalcaneal tendon modulus compared to 4-AP treatment (hypermotility)alone, and was statistically similar to controls (FIG. 5 ). BAPNtreatment reduced modulus. Notably, despite differences in tendonelastic modulus, apparent collagen organization appeared normal after4-AP, 4-AP+BAPN, and BAPN treatments.

TABLE 3 Average stage of chick embryos treated with saline, DMB, 4-AP,4-AP + BAPN, and BAPN at HH43 for 48 h (N ≥ 6). Expected stage after 48h treatment is HH45. Statistical analysis was performed to compareaverage stage of each group with saline control. Saline DMB 4-AP 4-AP +BAPN BAPN Stage (HH) of embryos at 45 ± 0.6 45 ± 0.4 45 ± 0.4 44 ± 0.545 ± 0.5 harvest (mean ± standard deviation (std)) p value (compared tosaline p > 0.05 p > 0.05 p > 0.05 *p < 0.05 p > 0.05 control)

Example 7 Treatment with Exogenous LOX Increased Elastic Modulus

In this example, assays were carried out to examine effects of exogenousLOX on elastic modulus of HH40 explant calcaneal tendons.

Briefly, HH40 explant calcaneal tendons were either cultured in a growthmedium supplemented with recombinant LOX (rLOX) (ORIGENE) or a saline(vehicle control) medium. The growth medium contained 10% fetal bovineserum and 1% antibiotic-antimycotic in high glucose Dulbecco's ModifiedEagle Medium (DMEM). Treatment of rLOX was performed at 0 h and 24 h.The growth medium were changed at 24 h. The explant tendons were thentested using tensile mechanical testing at 60 h. Two concentrations ofrLOX were used: 0.15 μg/uL and 1.5 μg/uL. The results are shown in FIG.7 .

As shown in FIG. 7 , the rLOX-treated explant tendons possessedsignificantly higher elastic modulus compared to saline-treated samplesafter 60 hours in culture. These results strongly indicate LOX as animportant player in enhancing mechanical properties of tendon.

Example 8 Effect of Rigid and Flaccid Paralysis on Calcaneal Tendon HPCrosslink Density Normalized to Collagen Content

In this example, assays were carried out to examine effects ofDMB-induced rigid paralysis and PB-induced flaccid paralysis on densityof collagen crosslinker hydroxylysyl pyridinoline.

Briefly, HH43 chick embryos were either treated with saline (vehiclecontrol), decamethonium bromide (DMB, THERMO FISHER), or pancuroniumbromide (PB, THERMO FISHER). Treatments of DMB and PB were performed at0 h and 24 h by injection through the air-pocket of each egg. After the48 h of treatments, each embryo was sacrificed by decapitation, wasstaged, and the calcaneal tendons were dissected for collagen cross-linkdensity analysis. For each biological sample (N=1), 5 tendons werepooled together (5 left calcaneal tendons from 5 chick embryos). In thisexperiment, collagen crosslink density analysis was performed forbiological samples. The results are shown in FIG. 8 . As shown in FIG. 8, both DMB- and PB-treated tendons possessed lower hydroxylysylpyridinoline (HP)/collagen ratio compared to the saline-treated tendonsafter 48 h of paralysis. The results indicate that paralysis induced byDMB treatment or PB treatment each decreased collagen cross-linking.

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The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated by reference in their entireties.

What is claimed is:
 1. A method (i) for improving a mechanical propertyof a tissue or a component thereof in a subject or (ii) for regeneratingan injured or diseased tissue or a component thereof in a subject or(iii) for developing embryonic/fetal tissue or a component thereof in asubject, comprising: (a) applying a mechanical stimulation to the tissueor cells therein, and (b) increasing the level of lysyl oxidase (LOX)activity in the tissue by delivering to the tissue a LOX polypeptide, apre-proLOX polypeptide, a proLOX polypeptide, a nucleic acid encodingone or more of the polypeptides, or a viral particle having the nucleicacid.
 2. The method of claim 1, wherein said mechanical stimulationcomprises one or more of a dynamic stimulation, a cyclic stimulation, astatic stimulation, a deformation, a tensile stimulation, a compressivestimulation, a torsion stimulation, a shear stimulation, substratestiffness, a mechanical loading, a static loading, a dynamic loading, acyclic loading, a compression, shear, torsion, and deformation.
 3. Themethod of claim 1, wherein the tissue is a natural tissue, an engineeredtissue, an embryonic tissue, a postnatal tissue, a tissue in vitro, or atissue in vivo.
 4. The method of claim 3, wherein the tissue is acollagenous or collagen-containing tissue.
 5. The method of claim 1,wherein the mechanical property is selected from the group consisting ofelastic modulus, tensile strength, torsional strength, elongation tobreak, hardness, compressive strength, burst strength, toughness, impactstrength, torsion, failure load, and stiffness.
 6. The method of claim1, wherein the LOX activity is an activity of a LOX polypeptide or aLOX-like (LOXL) polypeptide.
 7. The method of claim 1, furthercomprising administering a population of cells to the tissue.
 8. Themethod of claim 7, wherein the cells are (i) collagen-producing orelastin-producing cells or progenitor cells thereof or (ii) engineeredto release a specific factor that directly or indirectly promotesLOX/LOXL or pro-LOX/pro-LOXL gene expression, LOX/LOXL orpro-LOX/pro-LOXL protein expression, or LOX/LOXL enzyme activity.
 9. Themethod of claim 1, wherein this tissue is a tendon tissue or a ligamenttissue.
 10. The method of claim 1, wherein the tissue is a healthytissue.
 11. The method of claim 1, wherein the mechanical stimulationincreases the level of LOX activity in the tissue.